A deterministic, c-di-GMP-dependent program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations.

predicted by AlphaFold-Multimer.The model marked by a green box was used for further analysis.e, Bio-Layer Interferometric analysis of the binding kinetics of MalE-tagged DmxA WT to c-di-GMP.Streptavidin-coated sensors were loaded with 500 nM biotinylated c-di-GMP and probed with indicated concentrations of MalE-DmxA WT .The interaction kinetics were followed by monitoring the wavelength shifts during the association or dissociation of the analyte.Source data are provided as a Source Data file.(bottom).Protein was isolated from the same number of cells from the indicated strains grown on 1% CTT 1.5% agar plates.Protein from the same number of cells was loaded per lane.The blot for cellular PilA was stripped and then probed with α-LonD as a loading control.Calculated molecular weights of proteins are indicated.Numbers indicate the average level of PilA from n=3 biological replicates normalized to the loading control and relative to WT (100%).* p=0.0006 for ΔpilA vs WT; p=0.0245 for ΔdmxA vs WT, two-sided Student's t-test compared to WT. c, Determination of EPS synthesis.Cell suspensions of strains of the indicated genotypes were spotted on 0.5% agar supplemented with 0.5% CTT and Congo red or Trypan blue and incubated for 24 h.The ΔpilA mutant does not accumulate EPS 1 and was used as negative control.d-f Localization of the proteins of the polarity module (d) as well as schematics of the gliding motility complex (e) and the T4PM (f).In d, proteins of the polarity module are indicated [2][3][4][5][6][7][8][9][10] .Upon signaling by the Frz chemosensory system, their polarity is inverted.For each protein, the size of a circle indicates the amount of polarly localized protein.In e, proteins labeled with single letters in light blue, dark blue or brown have the Glt, Cgl or Agl prefix, respectively [11][12][13][14][15][16][17][18] .The AglR/-Q/-S complex harnesses proton-motive force to power gliding motility 19,20 .In f, proteins labeled with single letters have the Pil prefix.The pilus fiber is formed by PilA subunits and a tip complex, composed of PilY1 and four minor pilins (blue: PilX, orange: PilV, green: PilW, yellow: FimU) 21,22 .Tgl is an OM lipoprotein that is required for PilQ secretin assembly [23][24][25] .The ATPases PilB and PilT interact with the base of the T4PM in a mutually exclusive manner to stimulate extension and retraction of the pilus, respectively 21,26,27 .Bent arrows indicate incorporation of and removal of PilA subunits from the pilus base during extension and retraction, respectively.In e and f, lipoproteins are indicated by wavy black lines.g-i, Log2 fold-change of the accumulation of proteins of the Frz chemosensory system and the polarity module (g), the Glt/Agl complex (h), and the T4PM (i) in the ΔdmxA strain compared to WT. ND, not detected.Data points represent n=4 biological replicates.Error bars, mean±SD based on these four replicates.Source data are provided as a Source Data file.Cappable-seq as base-by-base alignment coverage for total RNA isolated from cells growing in 1% CTT broth 28 .Upper panel, genetic organization of dmxA locus as in c.Lower panel, positive and negative values indicate reads mapped to the forward and reverse strand, respectively.Reads assigned to a gene are colored according to the gene colour code in c; intergenic regions are in gray.Cappable-seq coverage is shown as black bars.e, Operon mapping of the dmxA locus.Upper panel, genetic organization of dmxA locus as in c.Letters and black lines below the genes indicate the fragments amplified by PCR (~400-500 bp).Bottom panel, the PCR products amplified using genomic DNA, cDNA, and an enzyme-free reverse transcription reaction (no RT) as templates were separated on a 1% agarose gel.Letters above the individual lanes correspond to the letters of the primer combinations depicted above.Molecular size markers in kilo base-pairs (kbp) are shown on the left.Representative image from n=2 biological replicates.f, Conservation of dmxA and its genetic neighborhood in other Myxococcales.Species included are listed in Table S6.Double slashes indicate no close proximity between the genes.g, Growth curve.Exponentially growing cells were diluted to an optical density (OD) at 550 nm (OD550) of 0.04 and growth was followed over time.Growth curves were generated from n=3 biological replicates.Error bars, mean±SD.h, Cell length determination.The cell length distribution of three biological replicates is shown in a violin plot.Each violin indicates the probability density of the data at different cell length values.Single points represent the median of the three biological replicates indicated in different colours (n=4956 from three biological replicates with each 1652 cells).Median is represented by a continuous red line.Dashed lines indicate 25 th and 75 th percentiles.Samples were compared using a two-sided Mann-Whitney test.Source data are provided as a Source Data file.cells from exponentially growing suspension cultures was loaded per lane.For the depletion of FtsZ, cells were exponentially grown in suspension culture in the presence of 10 µM vanillate for ~8 generations before the start of the experiment.Then, cells were washed and subsequently grown for 10 h without vanillate.The same blot was stripped before applying a new antibody.PilC served as a loading control.Calculated molecular weights of proteins are indicated.c, Immunoblot detection of DmxA-, FtsK-and sfGFP-miniTurbo.DmxA-miniTurbo-FLAG and FtsK-miniTurbo-FLAG were expressed from the pilA promoter (PpilA) and sfGFP-miniTurbo-FLAG from the Pvan in the presence of 100 µM vanillate.Protein from 7.00×10 7 cells from exponentially growing suspension cultures treated with or without biotin for 4 h was loaded per lane.The same blot was stripped before applying a new antibody.PilC served as a loading control.Western blots are representative from n=2 biological replicates.Source data are provided as a Source Data file.

Figure S1 .
Figure S1.In silico and in vitro analysis of DmxA variants.a, Calibration curve of SEC column.Molecular weight standards including their molecular mass are indicated in black and MalE-DmxA variants in grey dots.b, SEC of MalE-DmxA variants.Domain architectures of truncated DmxA variants are shown above chromatograms.Gray arrows indicate the void volume and black arrows the elution volume with the corresponding calculated molecular weight.c, Purified MalE-tagged DmxA variants after SEC.The MalE-tagged DmxA variants were separated by SDS-PAGE and stained with Coomassie blue.Calculated molecular weights of proteins are indicated.Molecular size markers are indicated on the left.d, pLDDT and pAE plots for five models of the DmxA dimer structure in Fig. 1c predicted by AlphaFold-Multimer.The model marked by a green box was

Figure S2 .
Figure S2.Phenotypic characterization and proteome analysis of the ΔdmxA mutant.a, Transmission electron microscopy of representative WT and a ΔdmxA cells with T4P at one pole.T4P are indicated by black arrowheads.The image in the middle shows the complete cell and the images on the left and right show the two poles.Scale bars, 0.5 µm (left and right), 2 µm (middle).b, T4P shear-off assay.Immunoblot detection of PilA in sheared T4P (top) and in total cell extract (bottom).Protein was isolated from the same number of cells from the indicated strains grown on 1% CTT 1.5% agar plates.Protein from the same number of cells was loaded per lane.The blot for cellular PilA was stripped and

Figure S3 .
Figure S3.Analysis of DmxA-mVenus accumulation and the dmxA locus.a, Distribution of the normalized cellular DmxA-mVenus fluorescence in single cells (n=1329 from three biological replicates, number of cells per replicate in brackets).Single cell total cellular fluorescence was normalized by the cell area to obtain the normalized cellular fluorescence.The distribution of the normalized cellular florescence is shown in histogram and was fitted to a Gaussian curve indicating a good fit to a unimodal distribution.b, Quantification of normalized cellular DmxA-mVenus fluorescence in time-lapse microscopy (n=23 division events from three biological replicates, number of events per replicate in brackets).DmxA-mVenus signals were determined before division in the predivisional cell (grey) and after division in the daughter cells (pink and yellow) at the indicated time points.Error bars, mean±SD.c, dmxA locus.Genes are drawn to scale and MXAN numbers or gene names are indicated.Predicted protein function is indicated.d, RNA-seq and

Figure S4 .
Figure S4.Accumulation of DmxA-mVenus, FtsZ and miniTurbo variants.a, Immunoblot detection of DmxA-mVenus.Protein from 5.60×10 8 cells from exponentially growing suspension cultures treated with cephalexin for 5 h as indicated was loaded per lane.The same blot was stripped before applying a new antibody.PilC served as a loading control.Calculated molecular weights of proteins are indicated.b, Immunoblot detection of FtsZ and DmxA-mVenus.Protein from 1.05×10 8 cells from exponentially growing suspension cultures was loaded per lane.For the depletion of FtsZ, cells were exponentially grown in suspension culture in the presence of 10 µM vanillate for ~8 generations before the start of the experiment.Then, cells were washed and subsequently grown for 10 h without vanillate.The same blot was stripped before applying a new antibody.PilC served as a loading

Figure S5 .
Figure S5.Accumulation of DmxA-mVenus variants and gliding in their presence.a, Immunoblot detection of DmxA-mVenus variants.Protein from 1.40×10 8 cells from exponentially growing suspension cultures was loaded per lane.The same blot was stripped before applying a new antibody.LonD served as a loading control.Calculated molecular weights of proteins are indicated.Representative blots from n=2 biological replicates.b, Gliding was analyzed on 1.5% agar.Scale bars, 1 mm (upper panels), 50 μm (lower panels).Source data are provided as a Source Data file.

Figure S6 .
Figure S6.Accumulation and analysis of cdGreen2 and mScarlet-I fluorescence a, Analysis of the cdGreen2 and mScarlet-I fluorescence in WT cells.White arrows indicate cells with bright cdGreen2 fluorescence.Scale bar, 10 μm.b, Immunoblot detection of cdGreen2 and mScarlet-I.Protein from 7.00×10 7 cells from exponentially growing suspension cultures was loaded per lane.The same blot was stripped before applying a new antibody.LonD served as a loading control.Calculated molecular weights of proteins are indicated.Representative blots from n=2 biological replicates.c, Localization of the DmxA-mVenus in Δ10 cells by epifluorescence microscopy.The mean±SD percentage of cells with a cluster at mid-cell (n=600 from three biological replicates with each 200 cells) is indicated.White arrowhead indicate cluster.Scale bar, 5 μm.d, cdGreen2 fluorescence in WT cells during the cell cycle.Epifluorescence and phase-contrast images from time-lapse microscopy of a representative cell.In every division event, a high cdGreen2 signal was observed (n=73 division events from three biological replicates, number of events per replicate in brackets).Images were recorded every 10 min; arrowhead, completion of cytokinesis.e-f, Analysis of representative WT cells expressing DmxA-mVenus (e) or cdGreen2 (f) treated with cephalexin.Images were recorded every 5 min (e) or every 10 min (f).g, c-di-GMP level of indicated strains during growth.The level of c-di-GMP is shown as the mean±SD from n=5 biological replicates.Individual data points are shown.Samples 8
* Fold change between the average intensities of the FtsK-miniTurbo-FLAG and the sfGFP-miniTurbo-FLAG samples.**Log10 of p-value:

Table S4 .
Plasmids used in this work.

Table S5 .
1ligonucleotides used in this work1

Table S6 .
Fully sequenced myxobacterial genomes used for the 16S rRNA tree